The overall goal of this proposal is to understand the role of altered intracellular Ca handling in the pathophysiology of heart failure. The proposal focuses on a recently discovered abnormality in the function of the sarcoplasmic reticulum (SR) Ca release channel (also known as the cardiac ryanodine receptor, RyR2) that makes the RyR2 overly active, i.e. "leaky" for Ca. In HF, the RyR2 becomes leaky due to abnormal modulation of the channel by Ca from inside the SR, a mechanism that normally operates to facilitate the transition of the RyR2s into a refractory state following SR Ca release. RyR2 luminal Ca regulation involves cooperation of several proteins associated with the RyR2 from the luminal side, including triadin 1, junctin and calsequestrin. Increased RyR2 activity would lead to Ca depletion in the SR, reducing Ca available for contraction, potentially contributing to the weakened cardiac contractile force in HF. Additionally, a sustained Ca leak might be capable of activating Ca dependent kinases and phosphatases that can feed back on RyR2s to cause more leak and a further derangement of the Ca release machinery. A comprehensive research plan is proposed to define the specific molecular causes responsible for this RyR2 dysfunction and its role in abnormal Ca handling of failing myocytes and in the natural development of HF. Our studies will use a unique combination of in vivo techniques and methods of cellular and molecular physiology, including patch clamp measurements, Ca imaging in the cytosolic and SR compartments and recording from single reconstituted RyR2 channels. We will use a large animal model of chronic HF which is highly relevant to human HF. The specific questions that will be addressed in this research proposal include: 1) What are the relative roles of altered SERCA2-mediated uptake, reduced NCX removal and enhanced SR Ca leak in altered Ca handling;2) Can the defective Ca homeostasis of failing myocytes be normalized by RyR2 inhibitors or by genetically targeting regulatory proteins involved in RyR2 luminal Ca-dependent modulation;3) Is abnormal RyR2 gating behavior is caused by abnormal phosphorylation/dephosphorylation of the RyR2 and/or by altered interactions with luminal auxiliary proteins involved in luminal Ca sensing;4) Is abnormal Ca handling a cause or a consequence of HF;and 5) Does transition to end stage HF involve an enhanced RyR leak, and do therapeutic interventions such as cardiac resynchronization therapy act by normalizing abnormal RyR2 function. RELEVANCE: Heart failure occurs when the heart is unable to pump enough blood to meet the needs of the body. Heart failure continues to increase in the U.S. population and is the most common diagnosis in hospitalized patients over the age of 65. We propose to study how abnormalities in the regulation of calcium in the heart muscle contribute to heart failure.